The present invention relates generally to portable, diagnostics, pocket-size incubators with integrated perfusion, and more particularly, to a biocompatible, disposable, centimeter-scale, incubator workstation 10 for in situ monitoring, handling and scientific studies of biological cultures in the field and within major laboratories.
Currently, the handling of biological samples intended for experimentation and diagnostic analysis is often limited by the need to carefully manage the conditions of perfusion and incubation, and to quickly return the samples to a controlled environment, especially in field situations. In many cases, necessary manipulation of samples for analytical purposes can result in unwanted contamination, lost time and considerable costs associated with repeat experiments.
Conventional incubators are relatively large devices, having a size that is comparable to a small bar refrigerator. Typically, static cultures are formed on Petri dishes and then placed in the incubator on shelves. While the incubator may typically have temperature, humidity and gas control, there is generally no provision for optically inspecting the cultures without opening the incubator and removing the Petri dish from the incubator. This exposes the culture to possible contamination, and may cause other problems relating to culture growth. Hence, it would be desirable to have an improved centimeter-scale, diagnostics incubator with integrated perfusion that may be used to culture biological samples with the better control of the cellular micro-environment. It would also be desirable to have a centimeter-scale, integrated diagnostics incubator that permits in-situ optical inspection of cultures within the incubator.
Experiments involving physiologically faithful, thick, three-dimensional (3-D) in-vitro cultures are time constrained as the tissue decays metabolically in the absence of functional vasculature and perfusion, often well before the relevant studies have been completed. Thus, it would be desirable to a have a centimeter scale diagnostic incubator with integrated perfusion that prevents high-density cultures from decaying metabolically by actively controlling the nutrient medium exchange rate and enabling the forced convection, intercellular mass transport to overcome diffusion limits in nutrient and gas delivery through the culture.
Since high-density cultures require many cells it would be desirable that the size of the cell culture chamber be as small as possible to reduce expenditures and enable high-plating densities with the low number of cells. It would be desirable that the centimeter-scale diagnostics incubator has small overall dimensions that would facilitate the control of the cellular microenvironment and provide the shortest possible response time to the physical changes and chemical stimuli. It would also be desirable to have a centimeter-scale, integrated diagnostics incubator that is portable and may be used in the field. Ultimately, it would also be desirable to have a centimeter-scale, integrated diagnostics incubator of a modular design to address different demands.
While 2-D culturing constitutes a standard practice for many fundamental studies, researchers are increasingly implementing the 3-D cell culture systems because they are biologically more realistic in capturing the in-vivo condition than their 2-D correlates. The three-dimensionality enables scientists to investigate cellular behavior in a more physiologically relevant state, while preserving the primary advantages of traditional in vitro systems, such as the control of cellular environment, accessibility for imaging, and elimination of systemic effects. Cells cultured in a 3-D environment are found to better represent the in vivo cellular behavior than cells cultured in monolayers. This was shown for different kinds of cell lines, e.g. for fibroblasts cells by Grinnell F. in 2000 Trends Cell Biol 10:362-365; for breast cells, e.g., Wang F. et al. in 1998 Proc Natl Acad Sci USA 95:14821-14826; for osteoblastic cells, by Granet C. et al. in 1998 Med Biol Eng Comput 36:513-519; and, neural cells by Fawcett J. W. et al. in 1989 Dev Biol 135:449-458, or Fawcett J. W. et al. in 1995 Exp Brain Res 106:275-282.
The way cells interact with each other and their microenvironment is fundamentally different in 3-D and 2-D cultures, see for example Schmeichel K. L. et al. in 2003, J Cell Sci 11:2377-2388. In many cases these interactions are reduced or negligible in 2-D cultures. This necessitated the development of neural cell culture models to support high, 3-D cellular densities with cells evenly distributed throughout the full thickness of the matrix. The extracellular matrix material supports the cells in a 3-D setting, and, enhances cell-to-cell and cell-to-matrix interactions, e.g., O'Connor S. M. et al. in 2001 Neurosci Lett 304:189-193; Woerly S. et al. in 1996 Neurosci Lett 205:197-201. However, these cultures have relied on passive diffusion for nutrient delivery and removal of toxic waste products necessitating the use of cell densities much lower than those found in the brain for example. Therefore, diffusion limited mass transport in nutrient delivery prevented the development of more in vivo resembling 3-D neural cell culture models having high cellular density (≧104 cells/mm3) and uniform cell distribution throughout culture thickness (>500 μm).
Growing demands for long-term incubation of physiologically faithful, three-dimensional (3-D) neuronal and other cultures during extended physiological studies require efficient perfusion platforms with functional vasculatures that mimic the in vivo condition in a thermally regulated environment. While expensive, relatively small, incubation baths with thermostatically controlled water jackets and capillary action perfusion are available commercially, to date, they remain incompatible with the microfabrication processes with their use confined to specific experimental conditions associated with the limits of capillary action perfusion. Representative incubation baths with passive perfusion are disclosed by Haas H. L. et al. in 1979 J Neurosci Meth 1:323-325, and, Zbicz K. L. et al. in 1985 J Neurophysiol 53:1038-1058.
The widespread use of 3-D neural cultures in medical research, however, is often hindered by low water solubility of oxygen, limited diffusion of media and oxygen through the tissue, and poor waste removal. Once the slices are being harvested, the utility of experiments is restricted by the quality of tissue perfusion, as the success of electrophysiological studies, for example, depends on long term slice viability to take reliable recordings.
There are generally two kinds of perfusion chambers that are being used to extend the viability of acute tissue slices in vitro based on constant circulation of the culture medium by passively augmenting the supply of media and oxygen. In the first kind, the tissue is submersed in the bathing solution and perfused using oxygenated media as discussed by Richards C. D. et al. in 1977 Br J Pharmacol 59:526P; Nicoll R. A. et al. in 1981 J Neurosci Meth 4:153-156; Palovcik R. A. et al. in 1986 J Neurosci Meth 17:129-139; Shi W. X. et al. in 1990 J Neurosci Meth 35:235-240, for example. In the second kind, the tissue rests on a mesh at the interface between the open channel flow of perfusate, underneath the mesh holding the tissue, and oxygenated and humidified atmosphere above the mesh. Representative chambers are described by Li C. L. et al. in 1957 J Physiol-London 139:178-190; Reynaud J. C. et al. in 1995 J Neurosci Meth. 58:203-208; Krimer L. S et al. in 1997 J Neurosci Meth 75:55-58.
Both approaches have practical benefits and shortcomings. The principal advantage of the submerged tissue incubation is faster diffusion of the bathing solution into the slice than in interface type chambers where only one side of the tissue is exposed to the media, not both. Although intuitively tissue would seem to have better oxygenation lying at the interface than being submerged, due to limited water solubility of oxygen in the latter, Croning M. D. R. et al. in 1998 J Neurosci Meth 81:103-11 found that the degree of disruption of ionic homeostatis by anoxia in rat hippocampal slices was greater when they were maintained at the interface. This could be attributed to the discontinuities in the flow of media associated with the surface tension effects at the gas-liquid interface. In addition to being simpler to design, submerged chambers provide more constant environment with less perturbations in fluid flow, purging of bubbles, and, draining, and, permit rapid exchanges of the bathing medium. Hence it appears that better perfusion fixation, in preventing both the starvation and anoxia during the process of observation, can be achieved in submerged chambers. Still, a major drawback in submerging the tissue below the liquid surface is actually keeping it submerged, because it will float unless it is properly attached to the perfusing substrate or restrained otherwise. In addition, localized injection of drugs is virtually impossible to administer with the current designs.
The dominant mode of mass transport in these scarcely used chambers remains diffusion and/or capillary action to passively augment the supply of media and oxygen to the tissue that eventually runs down metabolically. However, to meet and exceed the metabolic requirements for nutrient delivery and catabolic waste removal, mass transport by pure diffusion becomes insufficient and demands a convective enhancement for an adequate, dynamic, long-term control of the culture condition.